The small protein ubiquitin is involved in many crucial cellular processes in all eukaryotes. These include DNA repair, cell cycle control, programmed cell death, protein degradation, ribosome biogenesis, stress response, receptor structure, and the modification of histones. These responses depend upon covalent attachment of the C-terminus of ubiquitin to the alpha or epsilon amino groups of other proteins. The reverse reaction, cleavage of ubiquitin conjugates, is controlled by the class of enzymes known as ubiquitin-C-terminal hydrolases (UCHs). Despite the recognized importance of the ubiquitin system and the many different proteins involved, very few relevant structures have been determined. As yet, no UCH structure has been reported. The aim of this proposal is to further understanding of the mechanisms and specificities of UCHs by determination of the crystal structure of Yuh1, a 26kDa UCH from Saccharomyces cerevisiae. Toward this objective, crystals of Yuh1 have been grown and usable data have been collected to a resolution of 3.1 Angstroms. Weak diffraction is visible to a resolution of 2.8 Angstroms. The mechanism-based inhibitor ubiquitin aldehyde (Ubal) binds tightly to many different UCH enzymes, including Yuh1. In addition to that of the free enzyme, the structure of the Yuh1.Ubal complex will also be determined. This structure will be especially informative since Ubal contains almost all of the Yuh1 substrate specificity determinants, and the complex closely resembles the putative catalytic intermediate. Crystals of the Yuh1.Ubal complex have been grown and good data collected to a resolution of 2.9 Angstroms. Advantages to studying yeast UCHs include the availability of recombinant proteins and, in the longer term, the potential to relate mutant phenotypes with structural features of the enzymes. Although not a specific aim of this proposal, a long-term goal is the determination of crystal structures for several different UCHs with the expectation that their comparison will help to reveal the basis for their different specificities and functions. Another long-term goal is to design UCH variants that are able to cleave with enhanced or altered specificities. Yuh1 shares sequence similarity with PGP9.5, a UCH which is one of the most abundant proteins in mammalian brain. This may have relevance for a number of neurodegenerative diseases, such as Alzheimer's and Parkinson's, which are characterized by deposits such as neurofibrillary tangles that are rich in ubiquitin conjugates. The amino acid sequence around the active site cysteine of Yuh1 has some similarity to the putative active site region of the human oncoprotein Tre-2, which is also a UCH.